PHYSIOLOGICAL CHEMISTRY OF INTEGRIN FUNCTION

整合素是一个细胞粘附/信号分子家族， 在发育、创伤修复、血管生成、免疫中的重要作用， 和组织完整性。 调节特定的整合素是一种潜在的 强大的治疗靶点，用于多种疾病，包括癌症， 自身免疫 整联蛋白的一个显著特征是 在不改变蛋白质的情况下， 表达水平。 例如，整合素LFA-1最初是 对静息淋巴细胞无活性，但粘附活性迅速增强。 通过暴露于趋化因子或抗原而增加。 LFA-1的变化 对配体的亲和力不是调节的主要机制。 在 相反，越来越多的证据表明整合素与 细胞骨架很重要。我们的假设是整合素活性 由多步级联调节，主要步骤如下：1） 整联蛋白最初附着在细胞骨架上， 与配体的扩散限制反应，2）活化释放 整合素从细胞骨架约束增加配体结合，3） 配体结合诱导整联蛋白的构象变化，和4） 连接的整联蛋白结合细胞质因子， 机械性能，以增强整合素的二维亲和力， 配体相互作用 我们将检验这些假设， 整合素相互作用（键）的生理化学， 基于荧光的方法。 在目标1中，我们将确定 淋巴细胞活化的生物学重要模式影响LFA-1 在电池基板接触区域中接合。 在目标2中，我们将确定 不同淋巴细胞活化方式如何影响LFA-1侧链 流动性作为从细胞骨架释放的测定。 在目标3中， 将决定最有效的探测整合素的策略 通过外源性细胞质表达的细胞骨架相互作用 域

Integrins are a family of cell adhesion/signaling molecules that play an important role in development, wound repair, angiogenesis, immunity, and tissue integrity. Regulation of specific integrins is a potentially powerful therapeutic target for diverse diseases including cancer and autoimmunity. A remarkable characteristic of integrins is the degree of cellular regulation that can be achieved without altering protein expression levels. For example, the integrin LFA-1 is initially inactive on resting lymphocytes, but adhesion activity is rapidly increased by exposure to chemokines or antigen. Changes in LFA-1 affinity for ligand are not the primary mechanism for regulation. In contrast, there is mounting evidence that interactions of integrins with the cytoskeleton is important. Our hypothesis is that integrin activity is regulated by a multistep cascade with the following major steps: 1) the integrin is initially attached to the cytoskeleton to prevent diffusion limited reaction with ligands, 2) activation releases the integrin from cytoskeletal constraints to increase ligand binding, 3) ligand binding induces a conformational change in the integrin, and 4) the ligated integrin binds cytoplasmic factors that regulate local mechanical properties to enhance two dimensional affinity of integrin- ligand interaction. We will test these hypotheses by examining the physiological chemistry of integrin interactions (bonds) using a novel fluorescence based approach. In Aim 1 we will determine how different biologically important modes of lymphocyte activation affect LFA-1 engagement in cell substrate contact areas. In Aim 2 we will determine how different modes of lymphocyte activation affect LFA-1 lateral mobility as an assay for release from the cytoskeleton. In Aim 3 we will determine the most effective strategy to probe integrin cytoskeletal interactions through expression of exogenous cytoplasmic domain.